Protection of Tregs, Suppression of Th1 and Th17 Cells, and Amelioration of Experimental Allergic Encephalomyelitis by a Physically-Modified Saline

Abstract

In multiple sclerosis (MS) and other autoimmune diseases, the autoreactive T cells overcome the resistance provided by the regulatory T cells (Tregs) due to a decrease in the number of Foxp3-expressing Tregs. Therefore, upregulation and/or maintenance of Tregs during an autoimmune insult may have therapeutic efficacy in autoimmune diseases. Although several immunomodulatory drugs and molecules are available, most present significant side effects over long-term use. Here we have undertaken an innovative approach to upregulate Tregs and achieve immunomodulation. RNS60 is a 0.9% saline solution generated by subjecting normal saline to Taylor-Couette-Poiseuille (TCP) flow under elevated oxygen pressure. RNS60, but not NS (normal saline), RNS10.3 (TCP-modified saline without excess oxygen) and PNS60 (saline containing excess oxygen without TCP modification), was found to upregulate Foxp3 and enrich Tregs in MBP-primed T cells. Moreover, RNS60, but not NS, RNS10.3 and PNS60, inhibited the production of nitric oxide (NO) and the expression of iNOS in MBP-primed splenocytes. Incubation of the cells with an NO donor abrogated the RNS60-mediated upregulation of Foxp3. These results suggest that RNS60 boosts Tregs via suppression of NO production. Consistent to the suppressive activity of Tregs towards autoreactive T cells, RNS60, but not NS, RNS10.3, or PNS60, suppressed the differentiation of Th17 and Th1 cells and shifted the balance towards a Th2 response. Finally, RNS60 treatment exhibited immunomodulation and ameliorated adoptive transfer of experimental allergic encephalomyelitis, an animal model of MS, via Tregs. These results describe a novel immunomodulatory property of RNS60 and suggest its exploration for therapeutic intervention in MS and other autoimmune disorders.

Selective inhibition of CDK7 ameliorates experimental arthritis in mice

“…Splenocytes were isolated from the spleens of treatmentnaïve CIA mice [18]. Spleens were aseptically removed from eight mice in each group and washed with cold PBS and the tissues were minced.…”

“…Two-color flow cytometry was performed as described previously [18]. In brief, 1 9 10 6 splenocytes suspended in flow staining buffer were incubated at 4°C with FITClabeled anti-CD4 (eBioscience) for 30 min, washed and resuspended in fixation solution.…”

Daily Controlled Consumption of an Electrokinetically Modified Water Alters the Fatigue Response as a Result of Strenuous Resistance Exercise

“…Instead, both fluids are generated using a process involving TCP flow, which produces strong, controlled turbulence and cavitation events in the fluid. Research on RNS60 suggests that its bioactivity is based on the presence of nanobubblebased physical structures stabilized by an electrical doublelayer at the liquid/gas interface [8][9][10]. The exact nature of these nanostructures is under investigation and the descriptive term "charge-stabilized nanostructures" has been used to refer to these entities.…”

“…The group that consumed the EMW displayed lower blood levels of creatine kinase and high sensitivity CRP at 48 h and 96 h after exercise [18], thus indicating that less cell damage and inflammation occurred as a result of the exercise protocol. The bioactivity of RNS60 has been linked to effects on intracellular signaling pathways that influence cell stability and function [8][9][10]. Using a mouse model of Parkinson's disease, Khasnavis et al [8] showed that RNS60 improved locomotor function and reduced inflammation and neuronal degeneration in animals challenged with methyl-phenyltetrahydropyridine intoxication compared to a controlled saline.…”

“…Reducing the onset and extent of muscle fatigue and DOMS during and after resistance exercise and/or sports performance is a major concern for athletes and sports medicine professionals. Recently, a modified isotonic saline solution, RNS60, which is generated through a process involving Taylor-Couette-Poiseuille (TCP) flow, has shown potent anti-inflammatory and cytoprotective effects across different disease states including allergic asthma and neuroinflammation [8][9][10] and is currently in an FDAapproved phase I/IIa safety study in healthy volunteers as well as patients with mild to moderate asthma. RNS60 is generated in a rotor/stator device by subjecting normal saline to TCP flow under elevated oxygen pressure [9].…”